The present invention relates to methods of producing transgenic sunflower (Helianthus annuus L.) plants. Specifically, methods for Agrobacterium-mediated transformation of sunflower cotyledon cells, induction of transgenic shoots, and regeneration of fertile transgenic sunflower plants are disclosed.
The expanding field of biotechnology provides the tools for scientists to introduce important traits into a variety of plant species. New technologies make possible the production of commercially viable transgenic crops having significant economic impact on the agricultural industry. These advancements enable the creation of new crop germplasm containing novel traits. Such traits include improvements in the nutritional quality, insect resistance, disease resistance, and yield of many crops. Sunflower (Helianthus annuus L.) is one of the world's most important oil crops. Accordingly, much effort is continually directed toward the genetic engineering of this agronomically important crop species.
Genetic engineering of plants is essentially a two-step process: transformation and regeneration. First, plant cells are transformed, thereby introducing a nucleic acid sequence, which is typically integrated into the genome of the host cell. Second, a sexually competent plant is regenerated from the transformed cells. The transformation and regeneration processes preferably are complementary such that successfully transformed tissues are further capable of developing into competent whole plants.
Several methods, well known in the art, are available for introducing DNA into plant cells. Suitable methods include, but are not limited to, bacterial infection, binary bacterial artificial chromosome vectors, and direct delivery of DNA, e.g., via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA coated particles (reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991).
Many plants, including several important crop species, have been transformed using an Agrobacterium tumefaciens mediated transformation methodology. Agrobacterium-mediated transformation of sunflower has also been reported (Schrammeijer et al., Plant Cell Reports, 9: 55–60, 1990; EP 0 486 234). Transformed tissues reportedly included hypocotyls, apical meristems, and protoplasm.
Agrobacterium-mediated transformation of sunflower cotyledons has also been attempted. Utilizing cotyledons affords several advantages that other plant tissues do not. For instance, unlike many plant explants, minimal manipulation is required to prepare cotyledons for the transformation and regeneration processes. Also, source tissue is readily available in the form of mature seeds. In addition, cotyledons have demonstrated high potential for plant regeneration in several plant species (Sharma et al., Plant Sci., 66: 247–254, 1990; Mante et al., In Vitro Cell Dev. Biol., 25: 385–388, 1989). Furthermore, cotyledons can often give rise to shoots without an intervening callus stage. As a result, whole plants are obtained more rapidly and efficiently (Knittel etal., Plant Science, 73: 219–226, 1991).
Unfortunately, sunflower cotyledons have proven largely refractory to Agrobacterium-mediated transformation. Many of these attempts require extensive preparation of the cotyledons or additional equipment (such as a particle gun). Furthermore, in the few instances where successful transformation is reported, the transformed cotyledons have generally not been competent for induction of transgenic shoots. In some instances, chimeric shoots have been reported. However, there have not been reports to date of successful transformation of sunflower cotyledons with subsequent regeneration of fertile transgenic sunflower plants.
Ceriani et al. (Plant Cell Physiol. 33(2): 157–164, 1992) report the susceptibility of sunflower cotyledons to Agrobacterium tumefaciens infection. A low frequency of tumor-like growths is reported on the cotyledons after co-culture with Agrobacterium tumefaciens. Ceriani does not, however, attempt to initiate shoot formation or regenerate plants from the reported Agrobacterium-infected cotyledons.
Baker et al. (In Vitro Cell Dev. 31(3): 68A, 1995) describe the transformation of tissue explants from sunflower, including cotyledons, using micro-particles coated with Agrobacterium tumefaciens. The Agrobacterium is dried onto the micro-particles in a manner that maintains the viability of the bacteria. The explants treated according to this method reportedly formed chimeric shoots containing positively transformed regions.
Laparra et al. (Euphytica. 85: 63–74, 1995) describe transformation of several sunflower tissues, including cotyledon explants, via direct gene transfer, particle bombardment, and Agrobacterium infection. Regions of cotyledons were reportedly transformed by Agrobacterium infection. These transformed cotyledons, however, were incapable of regenerating into transgenic shoots. Laparra explains that “transformation occurs in the region, but not the cell type, competent for shoot regeneration.” Conversely, when a correct cell type was successfully transformed, this transformed region was ill suited to the regeneration of transgenic shoots.
Clearly, there is a need in the art for improved methods of producing transgenic sunflower plants via transformation and regeneration of sunflower cotyledon tissue.